AIDS Therapies

Combination Therapy

The most successful current antiviral therapy used to treat HIV infection is combination therapy. This rigorous regimen of three drugs, usually two reverse transcriptase inhibitors and a protease inhibitor, has been responsible for reports of "undetectable" viral loads (<200 viral RNAs/mL) in patients after two to three months of treatment (1). Taking high doses of these compounds used together usually is accompanied by harsh side effects, requiring much refining of the combinations and doses prescribed by the treating physician. However, by targeting different stages of the virus’s reproductive cycle with the drug combination, the virus’s ability to mutate and avoid the effects of the drugs is severely hampered, for multiple simultaneous productive mutations are required. The federal recommendations for antiretroviral combination therapy (shown at right) generally include one highly active protease inhibitor taken with two nucleoside reverse transcriptase inhibitors, by picking one from each column in the table (2).

For HIV-infected individuals, the recent antiretroviral combination drug therapies have been very successful at both suppressing the viral load in the bloodstream and maintaining immune system constitution (3,4). For the first time, there has been a substantial decline in AIDS incidence and mortality for the past two years in the United States (5). However, the total elimination of the HIV virus in infected individuals is, to date, an unaccomplished goal. Unfortunately, HIV-1 remains in latently infected cells for at least 30 months after the initiation of antiretroviral therapy (6). Even for HIV-infected patients whose viral load has been brought to undetectable levels with combination therapies, once the therapies are discontinued the viral load will rebound to pre-treatment levels (7). An especially troubling concern is the recent evidence suggesting that specific immunity to HIV-1, both cellular and humoral, wanes after effective drug therapy (8,9). Thus, under the combination therapy, the immune system could become complacent, and once a patient is taken off the combination therapy, the immune system is less prepared to fight the virus than before the treatment was begun.

The Reverse Transcriptase Inhibitors

There are two classes of reverse transcriptase (RT) inhibitors: nucleoside analogs such as AZT, ddI, ddC, d4T, and 3TC, and non-nucleoside inhibitors such as nevirapine and delavirdine. The commonly used RT inhibitors act at the HIV genomic RNA transcription stage of viral replication, and some are effective at crossing the blood-brain barrier. Viral drug resistance is the Achilles heel of the RT inhibitors; for example, resistance to 3TC occurs universally in patients using the drug for two months or longer, either alone or in a two-drug combination. Unfortunately, the newer non-nucleoside RT inhibitors are also rendered ineffective in a very short time by a single easily attainable mutation, greatly limiting their use.

What is reverse transcriptase?

Reverse transcriptase (RT) is an enzyme common to all retroviruses. Once a retrovirus infects a cell, RT copies the viral RNA genome into DNA for later integration by another enzyme into the genome of the host cell. This important early event in the retrovirus's lifecycle makes RT a good target for drug therapy.

More about RT and the HIV lifecycle

The Nucleoside Analogs

As you may have already learned from studying enzyme kinetics, enzyme inhibitors frequently mimic the substrate of an enzyme to compete with the enzyme's natural substrate for access to its active site. These are called competitive enzyme inhibitors. One of RT's substrates is the group of nucleoside triphosphates (NTPs) floating inside the cell, such as ATP, TTP, CTP, and GTP. The nucleoside analog RT inhibitors enter a cell, and are phosphorylated by the cell to yield nucleoside triphosphate analogs. A common misconception, then, is that because these analogs resemble RT's natural NTP substrates, they must work as competitive inhibitors...yet this is not so!

Let's take a closer look to understand this. By far, the most widely used nucleoside analog is 3'-azido-3'-deoxythymidine (AZT), which is a thymine nucleoside with an azide group in place of the 3'-hydroxyl group. Recall that when nucleic acids are polymerized into chains (such as RNA and DNA), they are connected from this 3' end to the next nucleotide's 5' end. In a patient treated with AZT, when the HIV RT begins copying its genomic RNA into DNA, every once in a while it will grab a phosphorylated AZT out of the infected cell's nucleotide pool for polymerization of the new DNA viral genome, mistaking it for a TTP. When RT incorporates the AZT in the growing DNA strand, the azido group terminates further polymerization because it has no 3'-OH to add any further residues. Therefore, AZT does not work to hamper HIV replication by inhibiting RT as a competitive inhibitor, but instead is a chain terminator, exactly similar to the dideoxy nucleoside Sanger reagents used in DNA sequencing.

Other nucleoside analogs include 2',3'-dideoxycytididine (ddC) and 2',3'-dideoxyinosine (ddI), which behave in a similar fashion to AZT.

The Non-nucleoside Inhibitors

The most common non-nucleoside inhibitor is nevirapine, which does not bind to the active site, but instead binds to a hydrophobic pocket elsewhere on the enzyme, inhibiting RT. The problem with non-nucleoside inhibitors is that the viral pool easily produces mutant strains in the treated patient. Because the inhibitor does not bind to the active site on RT, the amino acids near the place it does bind are not as well conserved, and mutations are more tolerated than near the active site. This is mainly because amino acids distal to the active site generally do not have the same functional importance to RT's enzymatic activity.

The Protease Inhibitors

The much newer antiviral protease inhibitors work to shut down viral packaging and maturation, but unfortunately, none of the approved protease inhibitors can cross the blood-brain barrier, and already resistance is becoming a problem.

What is protease?

The HIV protease (PR) is a proteolytic enzyme, meaning that it functions to digest proteins by hydrolyzing (breaking) peptide bonds in a protein. It works very similarly to the serine proteases trypsin and chymotrypsin that are used as examples in biochemistry courses, except that the HIV protease is an aspartic protease, containing a conserved aspartate amino acid in its active site instead of the serine residue common to the serine proteases.

When HIV proteins are being made from the integrated viral genome, they are produced as large polyprotein complexes that have to be proteolytically processed (digested) into the individual functional proteins by protease before a virion particle becomes fully infectious, or "mature." Because the integrity of digestion is related to how infectious new viral particles will be, the HIV protease also makes a promising antiviral therapeutic target because of its functional significance.

The Peptidomimetic Inhibitors

The inhibitors used to treat HIV infection by shutting down the viral protease (PR) strongly resemble PR's own natural substrate peptides, except that the PR inhibitors generally mimic the tetrahedral transition state for the reaction. If you recall, enzymes (such as PR) work by binding strongly to the transition state geometry of their substrate during the catalyzed reaction, thereby stabilizing the transition state and lowering the activation energy of the transition state with the contribution of the enzymes' own binding energy. So by emulating the transition state of the naturally catalyzed reaction in designing an inhibitor, one can produce an inhibitor that exhibits strong binding to the active site, which is the hallmark of a good competitive inhibitor.

More about protease and the HIV lifecycle

References

  1. Finzi, D., Hermankova, M., Pierson, T., Carruth, L. M., Buck, C., Chaisson, R. E., Quinn, T. C., Chadwick, K., Margolick, J., Brookmeyer, R., Gallant, J., Markowitz, M., Ho, D. D., Richman, D. D., Siliciano, R. F. 1997. Identification of a reservoir for HIV-1 patients on highly active antiretroviral therapy. Science 278: 1295-1300.
  2. Project Inform. 1998.
  3. Haase, A. T., Henry, K., Zupancic, M., Sedgewick, G., Faust, R. A., Melroe, H., Cavert, W., Gebhard, K., Staskus, K., Zhang, Z. Q., Dailey, P. J., Balfour, H. H. Jr, Erice, A., Perelson, A. S. 1996. Quantitative image analysis of HIV-1 infection in lymphoid tissue. Science 274: 985-989.
  4. Cavert, W., Notermans, D. W., Staskus, K., Wietgrefe, S. W., Zupancic, M., Gebhard, K., Henry, K., Zhang, Z. Q., Mills, R., McDade, H., Goudsmit, J., Danner, S. A., Haase, A. T. 1997. Kinetics of response in lymphoid tissues to antiretroviral therapy of HIV-1 infection. Science 276: 960.
  5. Palella, F. J., Delaney, K. M., Moorman, A. C., Loveless, M. O., Fuhrer, J., Satten, G. A., Aschman, R., Holmberg, S. D. 1998. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N. Engl. J. Med. 338: 853-860.
  6. Chun, T. W., Stuyver, L., Mizell, S. B., Ehler, L. A., Mican, J. A., Baseler, M., Lloyd, A. L., Nowak, M. A., Fauci, A. S. 1997. Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl. Acad. Sci. U.S.A. 94: 13193-13197.
  7. Wong, J. K., Gunthard, H. F., Havlir, D. V., Zhang, Z. Q., Haase, A. T., Ignacio, C. C., Kwok, S., Emini, E., Richman, D. D. 1997. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Proc. Natl. Acad. Sci. U.S.A. 94: 12574-12579.
  8. Ogg, G. S., Jin, X., Bonhoeffer, S., Dunbar, P. R., Nowak, M. A., Monard, S., Segal, J. P., Cao, Y., Rowland-Jones, S. L., Cerundolo, V., Hurley, A., Markowitz, M., Ho, D. D., Nixon, D. F., McMichael, A. J. 1998. Quantitation of HIV-1-specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 279: 2103-2106.
  9. Morris, L., Binley, J. M., Clas, B. A., Bonhoeffer, S., Astill, T. P., Kost, R., Hurley, A., Cao, Y., Markowitz, M., Ho, D. D., Moore, J. P. 1998. HIV-1 antigen-specific and -nonspecific B cell responses are sensitive to combination antiretroviral therapy. J. Exp. Med. 188: 233-245.

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